Composition and method for affecting metallocorrinoid uptake

ABSTRACT

The present invention is directed to compositions and methods for affecting metallocorrinoid uptake. The compositions and methods of the present invention are particularly useful in enhancing the uptake or availability of biologically active metallocorrinoids (e.g. cobalamin and its analogs). The present invention is particularly useful in the treatment or prevention of conditions that result from low expression or activity of proteins involved in the processing of metallocorrinoids, as well as in conditions which would benefit from enhanced uptake or availability of cobalamin or its biologically active analogs of cobalamin (e.g. cobalamin drug conjugates).

BACKGROUND OF THE INVENTION

[0001] Metallocorrinoids are corrin rings with a metal-atom center, suchas Co, Fe, Ni, or Mn. A corrin ring is four reduced pyrrole rings linkedtogether. A subclass of naturally occurring metallocorrinoids is knownas cobalamin, that is, a cobalt-centered corrin ring. Naturallyoccurring vitamin B₁₂, for example, is a cobalamin.

[0002] Vitamin B₁₂ compounds are known to have many biologicalfunctions. They are required by the enzyme methionine synthase, forexample, which is involved in the production of DNA. Pregnant women needincreased amounts of vitamin B₁₂ which is involved in the production ofred blood cells. It is also believed that vitamin B₁₂ enhances theeffects of other vitamins and nutrients in tissue repair. Lack ofvitamin B₁₂ leads to megaloblastic anemia (characterized by large andimmature red blood cells) and neuropathy in man with insidious onset ofsymptoms. These symptoms include weakness, tiredness, breathlessness(dyspnea) on exertion, tingling and numbness (paresthesia), sore tongue(glossitis), loss of appetite and weight, loss of sense of taste andsmell, impotence, psychiatric disturbances (such as irritability, memoryimpairment, mild depression, hallucinations) and severe anemia (whichmay lead to signs of cardiac dysfunction). Deficiency of vitamin B₁₂leads to defective DNA synthesis in cells; tissues most affected arethose with the greatest rate of cell turnover, e.g. the haematopoieticsystem. In small children Cbl deficiency can result in developmentaldelay, hematological disorders, and neurological disorders. There may beirreversible damage to the nervous system with specific demyelination ofthe spinal cord.

[0003] Increased availability of vitamin B₁₂, on the other hand, appearsto have a very beneficial effect. Cbl analogs and cobalamin drugconjugates have been shown to inhibit the growth of leukemia cells bypossibly deactivating methionine synthase, thus preventing DNAsynthesis. The cobalamins that are analogous to vitamin B₁₂ compoundswould appear to be potential therapeutic agents. These includehydroxocobalamin, cyanocobalamin, nitrocobalamin, mehtylcobalamin, and5′-deoxyadenocobalamin, as well as nitrosylcobalamin.

[0004] All forms of vitamin B₁₂ (adenosyl-, cyano-, hydroxo-, ormethylcobalamin) are bound by the transport proteins intrinsic factorand transcobalamin II, to be biologically active. Those transportproteins involved in the uptake of vitamin B₁₂ are referred to herein ascobalamin binding proteins. Specifically, gastrointestinal absorption ofvitamin B₁₂ relies upon the intrinsic factor-vitamin B₁₂ complex beingbound by the intrinsic factor receptors in the terminal ileum. Likewise,intravascular transport and subsequent cellular uptake of vitamin B₁₂throughout the body is dependent upon transcobalamin II and the cellmembrane transcobalamin II receptors, respectively. After thetranscobalamin II-vitamin B₁₂ complex has been internalized, thetransport protein undergoes lysozymal degradation, which releasesvitamin B₁₂ into the cytoplasm.

[0005] Cellular utilization of Cbl is preceded by two importantreceptor-mediated endocytic events. First, the dietary Cbl bound togastric intrinsic factor (IF), a 50-kDa glycoprotein, is transportedacross the absorptive enterocyte via an intrinsic factor-cobalaminreceptor that is expressed exclusively in the apical or the luminalmembranes. The plasma transport of cobalamin to tissues/cells appears tooccur via transporter transcobalamin II (TC II), by receptor-mediatedendocytosis via transcobalamin II-receptor (TC II-R). Intracellularlyreleased Cbl is then converted to its biologically active forms, (e.g.methyl-Cbl and 5′-deoxyadenosyl-Cbl) which are utilized by thecytoplasmic enzyme methionine synthase (MS) and mitochondrial enzymemethyl-malonyl-CoA mutase (MMCM), respectively. MS activity is requiredfor folate metabolism and DNA synthesis and presents a promising targetto block cell proliferation. TCII and serum Cbl levels are bothincreased in hepatocarcinomas and leukemias. TCII has been identified asan acute phase reactant in autoimmune disorders and infection. Severalstudies have shown that high levels of Cbl inhibited L1210, P388D1,CCRF-CEM, and NCTC929 cell proliferation. This is likely due to theactivation of an autoimmune response.

[0006] Recent studies have shown that TC II-R is expressed as anon-covalent homodimer of molecular mass of 124 kDa in tissue plasmamembranes of human, rat, and rabbit. A comprehensive review oftranscobalamin II, the transcobalamin II receptor, and the uptake ofvitamin B₁₂ is provided in “Transcobalamin II and Its Cell SurfaceReceptor Vitamins and Hormones”, Vitamins and Hormones, Vol. 59, pgs.337-366 (2000) which is incorporated herein in its entirety by referencethereto. Plasma membrane expression of TC II-R appears important for thetissue/cellular uptake of Cbl since its functional inactivation in vivoby its circulatory antiserum results in intracellular deficiency of Cbl.This intracellular deficiency in Cbl results in the development of Cbldeficiency of the animal as a whole.

[0007] The utilization of vitamin B₁₂ as a delivery vehicle is knownart. The art describes an oral delivery system that delivers activesubstances (hormones, bio-active peptides or therapeutic agents) bybinding these agents to cobalamin or an analog thereof

[0008] U.S. Pat. No. 5,936,082, which is hereby incorporated byreference in its entirety, for example, describes the therapeuticeffectiveness of vitamin B₁₂ based compounds. Nitrosylcobalamin(NO-Cbl), in particular, was evaluated for its chemotherapeutic effect.In five human hematological and eight solid tumor cell lines, NO-Cblexhibited an ID₅₀ that was 5-100 fold lower in tumor cell lines comparedto benign cells (fibroblasts and endothelial cells). When oxidized fromNO-Cbl, the NO free radical functions in a number of capacities. NO isinvolved in vasodilation, and is known to contribute to increasedoxidative stress, inhibition of cellular metabolism and induction of DNAdamage leading to apoptosis and/or necrosis.

[0009] Radiolabelled vitamin B₁₂ analogs have also been described in theart as useful in vivo imaging agents. For example, U.S. Pat. No.6,096,290, which is hereby incorporated herein in its entirety byreference thereto, describes the use of radiolabelled vitamin B₁₂analogs as in vivo tumor imaging agents.

[0010] U.S. Pat. No. 6,183,723, which is also incorporated herein byreference in its entirety, describes certain other cobalamin-drugconjugates.

SUMMARY OF THE INVENTION

[0011] The multiple components of Cbl uptake, enzymes, co-factors, andtransport systems present several points of attack for the therapeuticdelivery of cobalamins. As is described herein, the interrelationship ofTCII-R and cytokines make this an attractive target for the therapeuticdelivery of biologically active metallocorrinoids. Cytokines, inparticular interferon β, are shown to enhance the uptake or activity ofbiologically active metallocorrinoids, including vitamin B₁₂ analogs,homologs, and derivatives.

[0012] Vitamin B₁₂ analogs can be synthesized in a number of ways. Inaddition to conjugation of the side chains of the corrin ring,conjugation to the Cbl moiety can also be made, as can conjugation tothe ribose moiety, phosphate moiety, and to the benzimidazole moiety.The conjugating agent and the drug to be conjugated depend upon the typeof Cbl group that is modified and the nature of the drug. One of skillin the art would understand how to adapt the conjugation method to theparticular Cbl group and drug to be coupled.

[0013] Preferred methods of attaching the drug to the Cbl moleculeinclude conjugation to Cbl via biotin. Biotin is conjugated to eitherthe propionamide or the acetamide side chains of the corrin ring of theCbl molecule. The initial biotin-Cbl complex can be prepared accordingto Pathre, et al. (Pathre, P. M., et al., “Synthesis of Cobalamin-Biotinconjugates that vary in the position in cobalamin coupling, Evaluationof cobalamin derivative binding to transcobalamin II,” incorporated byreference). Vitamin B₁₂ is commercially available in its most stableform as cyanocobalamin from Sigma Chemical (St. Louis, Mo.).

[0014] One may most easily obtain transcobalamin II in the followingmanner: transcobalamin II cDNA is available in the laboratories of Drs.Seetharam (Medical College of Wisconsin) and Rothenberg (VA-Hospital,New York) TC II cDNA can be expressed in a Baculovirus system to make alarge amount of functionally active TC II protein (see Quadros, E. V.,et al., Blood 81:1239-1245, 1993). One of skill in the art would be ableto reproduce the TC II cDNA. The antibodies to TCII-R were obtainedthrough the laboratory of Dr. Bellur Seetharam, Med. College of WI.

[0015] One way to make cobalamin drug conjugates is through geneticengineering. In this method, a DNA sequence encoding TC II and thepeptide drug may be expressed as one chimeric molecule. For example, itis possible to generate a chimeric construct using the full-length TC IIcDNA and the cDNA for a peptide drug (e.g. insulin). The chimericconstruct can then be expressed to produce a fusion protein consistingof the TC II-peptide drug. Following synthesis, the chimeric proteinshould be tested for both TC II activity and drug activity. Cobalamincan then be allowed to bind to this chimeric protein and used fortherapy.

[0016] The observation that a cytokine (i.e. an interferon such asinterferon-β) upregulates or enhances the activity of the TCII-Rprovides a basis for a number of embodiments of the present invention.

[0017] One embodiment of the present invention is a method forincreasing cobalamin-binding protein activity in a subject in order totreat a condition favorably affected by an increase in saidcobalamin-binding activity, said method comprising the step ofadministering to a subject in need of such treatment a cytokine in anamount effective to increase cobalamin-binding activity in the subject.This method may further include the step of administering a vitamin B₁₂analog (which may be a naturally occurring vitamin B₁₂ analog),nitrosylcobalamin or other suitable vitamin B₁₂ drug conjugate. In thisembodiment, the cytokine may be administered prior, simultaneously, orconsecutively with the vitamin B₁₂ analog. The cytokine and/or vitaminB₁₂ analog may be administered prophylactically or acutely. Theincreased cobalamin binding protein activity is preferably TCII-Ractivity. The cytokine is preferably an interferon such as interferon-β.

[0018] Another embodiment of the present invention is a composition thatis comprised of a metallocorrinoid and a cytokine. It is preferable thatthe metallocorrinoid be a vitamin B₁₂ analog, homolog, derivative orsimply vitamin B₁₂. This is particularly. useful when there is adeficiency in vitamin B₁₂ or if the vitamin B₁₂ analog includes a drugconjugated thereto. It is particularly preferable that the vitamin B₁₂analog be a nitrosylcobalamin, but it may also be others known in theart, (e.g. hydroxocobalamin, cyanocobalamin, and methylcobalamin and5′deoxyadenocobalamin or radiolabelled cobalamin derivatives). Thecomposition in accordance with this embodiment of the invention may alsoinclude a pharmaceutical carrier. It is preferable that the cytokine bean interferon, and more particularly interferon-β.

[0019] Another embodiment of the present invention is a therapeuticcomposition comprising a cobalamin or cobalamin drug conjugate and acytokine such as interferon-β. In this embodiment, the therapeuticcomposition may also further comprise a pharmaceutical carrier. This isa particular advantageous embodiment when the cobalamin drug conjugateis designed for a specific aim in mind. Nitrosylcobalamin is just onecobalamin drug conjugate, and other drug conjugates may be selected fromthe group consisting of hydroxocobalamin, cyanocobalamin,methylcobalamin, and 5′deoxyadenocobalamin, radiolabelled cobalamin, orother cobalamin and drug conjugate. This embodiment is particular usefulin the treatment of diseases where the delivery of a therapeutic agentvia a cobalamin delivery mechanism would be beneficial.

[0020] Another embodiment of the present invention is a method ofenhancing uptake or activity of a metallocorrinoid comprised ofadministering a cytokine. It is preferable that the metallocorrinoid bea vitamin B₁₂ or a vitamin B₁₂ analog, homolog, or derivative. In thismethod it is preferable that the cytokine is an interferon, and morepreferably that the interferon be interferon-β.

[0021] Another embodiment of the present invention is a method ofenhancing cellular uptake of a metallocorrinoid comprising the step ofcontacting a cell with a cytokine, particularly where the step ofcontacting a cell with a cytokine occurs through induction of cytokine.In this embodiment, it is preferable that the metallocorrinoid isvitamin B₁₂ or a vitamin B₁₂ analog. As in other embodiments, thevitamin B₁₂ analog may be any suitable vitamin B₁₂ analog, homolog orderivatives such as a cobalamin drug conjugate. In this embodiment it ispreferable that the cytokine is an interferon, particularlyinterferon-β.

[0022] Another embodiment of the present invention is a method oftreating a patient comprising the steps of inducing cytokine production;and administering a metallocorrinoid. The step of inducing cytokineproduction may include administering a cytokine, or administering anagent as is known in the art to stimulate cytokine expression orproduction. The metallocorrinoid of this embodiment may be vitamin B₁₂or a vitamin B₁₂ analog, homolog or derivative such as a cobalamin drugconjugate. The cytokine is preferably an interferon, more preferablyinterferon-β.

[0023] Yet another embodiment of the present invention is a method ofenhancing bio-availability of a metallocorrinoid, comprising the step ofadministering interferon-β alone or in combination with ametallocorrinoid.

[0024] Yet another embodiment of the present invention is a method oftreating a subject to increase TCII-R activity in a cell comprising thestep of administering to a subject in need of such treatment a cytokineto increase TCII-R activity in an amount effective to increase TCII-Ractivity in said cell. In this embodiment, it is preferable that thesubject be cobalamin deficient. Another application of this embodimentis wherein the amount is sufficient to increase TCII-R activity abovenormal baseline levels. Preferably, this method may also be useful whenthe subject has an abnormally low level of TCII-R activity. This methodpreferably includes the step of co-administering a substrate (or ligand)of TCII-R, wherein the substrate of TCII-R is a cobalamin based compound(e.g. cobalamin or a cobalamin drug conjugate). The cobalamin drugconjugate is preferably nitrosylcobalamin, but may be any suitablecobalamin drug conjugate such as those known in the art.

[0025] Yet another embodiment of the present invention is a method oftreating cancer comprised of administering a cytokine (e.g. interferon βto enhance the uptake or increase the availability of cobalamin analogs,homologs, or derivatives. This can be done either alone or incombination with the cobalamin analog, homolog, or derivative.

[0026] Another embodiment of the present invention is a method ofimaging tissue or cells through enhanced uptake of radiolabelled vitaminB₁₂ analogs, homologs or derivatives via administration of a cytokinesuch as interferon β.

[0027] Additional aspects and applications of the present invention willbecome apparent to the skilled artisan upon consideration of thedetailed description of the invention, which follows.

BRIEF DESCRIPTION OF THE FIGURES

[0028] The file of this patent contains at least one drawing executed incolor. Copies of this patent with color drawing(s) will be provided bythe Patent and Trademark Office upon request and payment of thenecessary fee.

[0029]FIG. 1 is a bar graph illustrating the anti-proliferative effectof a cytokine (i.e. an interferon) and NO-Cbl on NIH-OVCAR-3 ovariancarcinoma;

[0030]FIG. 2 is a graph illustrating a median effect analysis inaccordance with the present invention;

[0031]FIG. 3 is a western blot analysis performed on extracts fromovarian carcinomas according to the present invention;

[0032]FIG. 4 illustrates a bar graph of a flow cytometric analysis ofAnnexin V positive cells;

[0033]FIG. 5 are stained cells illustrating up-regulated TCII-R incontrol and IFN-β in NIH-OVCAR-3 treated samples;

[0034]FIG. 6 is a bar graph illustrating the anti-proliferative effectof a cytokine (i.e. interferon) and NO-Cbl on WM9 melanoma;

[0035]FIG. 7 is a graph illustrating a median effect analysis-on WM9human melanoma cells;

[0036]FIG. 8 depicts treated and untreated WM9 tumor cells in accordancewith the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0037] While not wishing to be bound by theory, it appears that theuptake of metallocorrinoids such as vitamin B₁₂, NO-Cbl or other vitaminB₁₂-based compounds, is dependent upon the TCII receptor, specific forvitamin B₁₂. Because the TCII-R plays a central role in determiningmetallocorrinoid activity, the relationship between TCII-R and cytokines(e.g. interferons (“IFNs”)) was evaluated. IFNs upregulate theexpression of cell surface markers HLA-I, HLA-II, β2 microglobulin, andtumor associated antigens such as CEA and CA125.

[0038] The present invention provides for an increase in receptor orreceptor activity responsible for the uptake of vitamin B₁₂ derivedcompounds. The administration of cytokines, particularly interferonssuch as IFN-β, appears to enhance the activity of TCII-R. Administeringthese cytokines prior to or concurrently with vitamin B₁₂-basedcompounds increases the delivery of the vitamin B₁₂-based compounds andlike metallocorrinoids.

[0039] Increased activity (e.g. TCII-R activity) can be accomplished ina number of different ways. For example, an increase in the amount ofprotein or an increase in the activity of the protein (while maintaininga constant level of the protein) can result in increased “activity”. Anincrease in the amount of protein available can result from increasedtranscription of the gene, increased stability of the mRNA or a decreasein protein degradation.

[0040] The present invention, by causing an increase in Cbl-binding(e.g. TCII-R) activity, permits not only the re-establishment of normalbase-line levels of Cbl-binding activity, but also allows increasingsuch activity above normal base-line levels. Normal base-line levels arethe amounts of activity in a normal control group, controlled for ageand having no symptoms that would indicate alteration of Cbl-bindingactivity. The actual base line level will depend upon the particular agegroup selected and the particular measure employed to assay. When usingthe cytokines of the present invention not only can normal base-linelevels be restored, but abnormal activity can also be increaseddesirably far above normal base-line levels of TCII-R binding activity.Thus, “increasing activity” means any increase in Cbl-binding protein orcobalamin uptake in the subject resulting from the treatment, accordingto the invention, including, but not limited to, such activity as wouldbe sufficient to restore normal base-line levels, and such activity aswould be sufficient to elevate the activity above normal base-linelevels.

[0041] In one embodiment of the invention the increase in activity ofthe Cbl-binding activity is cytokine induced. Cytokines are solublepolypeptides produced by a wide variety of cells. Cytokines control geneactivation and cell surface molecule expression. In what follows, theterm “cytokine” incorporates families of endogenous molecules of variousdenominations: lymphokines, monokines, interleukins, interferons,colonization factors and growth factors and peptides. The knowncytokines are in particular interferon-α (IFN-α), interferon-β (IFN-β),γ-interferon (γ-IFN), interleukin-1 (IL-1) in α and β forms,interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4),interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-10 (IL-10),interleukin-12 (IL-12), tumor necrosis factor (TNF) in α and β forms,transforming growth factors (TGF-β), in β1, β2, ↑3, β1.2 forms, andcolony-stimulating factors (CSF) such as the granulocytemacrophage-stimulating factor (GM-CSF), the granulocytecolony-stimulating factor (G-CSF) and the macrophage-stimulating factor(M-CSF) and the epithelial growth factor (EGF), somatostatin,endorphins, the various “releasing factors” or “inhibitory factors” suchas TRF. There also exist pegilated forms of interferon. Cytokines playan essential role in the development of the immune system and thus inthe development of an immune response. However, besides their numerousbeneficial properties, they have also been implicated in the mechanismsfor the development of a variety of inflammatory diseases. For example,the cytokines TNF-α and IL-1 are thought to be part of the diseasecausing mechanism of atherosclerosis, transplant arteriosclerosis,rheumatoid arthritis, lupus, scleroderma, emphysema, etc.

[0042] Important embodiments of the invention involve populations neverbefore treated with a cytokine such as interferon. Thus, the inventioninvolves, in certain aspects, treatments of individuals who areotherwise free of symptoms calling for treatment with interferons.

[0043] The cytokines and/or cobalamin compounds are preferablyadministered in effective amounts. In general, an effective amount isany amount that can cause an increase in Cbl-binding proteins activityin a desired cell population or tissue, and preferably in an amountsufficient to cause a favorable phenotypic change in the condition suchas a lessening, alleviation or elimination of a symptom or of acondition.

[0044] With regard to the cobalamin or vitamin B₁₂ derived compounds, aneffective amount is that amount of a preparation that alone, or togetherwith further doses, produces the desired response. This may involve onlyslowing the progression of the disease temporarily, although morepreferably, it involves halting the progression of the diseasepermanently or delaying the onset of or preventing the disease orcondition from occurring. This can be monitored by routine methods.Generally, doses of active compounds would be from about 0.01 mg/kg perday to 1000 mg/kg per day. It is expected that doses ranging from 50-500mg/kg will be suitable, preferably intravenously, intramuscularly, orintradermally, and in one or several administrations per day.

[0045] Such amounts will depend, of course, on the particular conditionbeing treated, the severity of the condition and the individual patientparameters. Some parameters for consideration include age, physicalcondition, size and weight, the duration of the treatment, the nature ofconcurrent therapy (if any), the specific route of administration andlike factors within the knowledge and expertise of the healthpractitioner.

[0046] Intravenous administration and intramuscular administrationavoids transport problems associated with cobalamin when administeredorally. However, if the vitamin B₁₂ analog, homolog or derivative isencapsulated, oral delivery may be preferred. In the event that aresponse in a subject is insufficient at the initial doses applied,higher doses (or effectively higher doses by a different, more localizeddelivery route) may be employed to the extent that patient tolerancepermits. Multiple doses per day are contemplated to achieve appropriatesystemic levels of compounds. It is preferred generally that a maximumdose be used, that is, the highest safe dose according to sound medicaljudgment. Those of ordinary skill in the art will understand, however,that a patient may insist upon a lower dose or tolerable dose formedical reasons, psychological reasons or for virtually any otherreason.

[0047] The cytokines (e.g. interferons) useful according to theinvention may be combined, optionally, with apharmaceutically-acceptable carrier. The term“pharmaceutically-acceptable carrier” as used herein means one or morecompatible solid or liquid fillers, diluents or encapsulating substanceswhich are suitable for administration into a human. The term “carrier”denotes an organic or inorganic ingredient, natural or synthetic, withwhich the active ingredient is combined to facilitate the application.The components of the pharmaceutical compositions also are capable ofbeing co-mingled with the molecules of the present invention, and witheach other, in a manner such that there is no interaction which wouldsubstantially impair the desired pharmaceutical efficacy.

[0048] The pharmaceutical compositions may contain suitable bufferingagents, including: acetic acid in a salt; citric acid in a salt; boricacid in a salt; and phosphoric acid in a salt. The pharmaceuticalcompositions also may contain, optionally, suitable preservatives, suchas: benzalkonium chloride, chlorobutanol, parabens and thimerosal.

[0049] A variety of administration routes are available. The particularmode selected will depend, of course, upon the particular drug selected,the severity of the condition being treated and the dosage required fortherapeutic efficacy. The methods of the invention, generally speaking,may be practiced using any mode of administration that is medicallyacceptable, meaning any mode that produces effective levels of theactive compounds without causing clinically unacceptable adverseeffects. Such modes of administration include oral, rectal, topical,nasal, intradermal, inhalation, intra-peritoneal, or parenteral routes.The term “parenteral” includes subcutaneous, intravenous, intramuscular,or infusion. Intravenous or intramuscular routes are particularlysuitable for purposes of the present invention.

[0050] The pharmaceutical compositions may conveniently be presented inunit dosage form and may be prepared by any of the methods well known inthe art of pharmacy. All methods include the step of bringing the activeagent into association with a carrier that constitutes one or moreaccessory ingredients. In general, the compositions are prepared byuniformly and intimately bringing the active compound into associationwith a liquid carrier, a finely divided solid carrier, or both, andthen, if necessary, shaping the product.

[0051] Compositions suitable for parenteral administration convenientlycomprise a sterile aqueous preparation of the cytokines and/orcobalamins, which is preferably isotonic with the blood of therecipient. This aqueous preparation may be formulated according to knownmethods using suitable dispersing or wetting agents and suspendingagents. The sterile injectable preparation also may be a sterileinjectable solution or suspension in a non-toxic parenterally-acceptablediluent or solvent, for example, as a solution in 1,3-butane diol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose any bland fixed oil may be employedincluding synthetic mono-or di-glycerides. In addition, fatty acids suchas oleic acid may be used in the preparation of injectable's. Carrierformulation suitable for oral, subcutaneous, intravenous, intramuscular,etc. administrations can be found in Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa. which is incorporated hereinin its entirety by reference thereto.

[0052] Other delivery systems can include time-released, delayed releaseor sustained release delivery systems. Such systems can avoid repeatedadministrations of the active compound, increasing convenience to thesubject and the physician, and may be particularly suitable for certaincobalamin drug conjugates of the present invention, particularly thenitrosylcobalamin due to its activation under acidic conditions found inthe early gastrointestinal tract. Many types of release delivery systemsare available and known to those of ordinary skill in the art. Theyinclude polymer base systems such as poly(lactide-glycolide),copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters,polyhydroxybutyric acid, and polyanhydrides. Microcapsules of theforegoing polymers containing drugs are described in, for example, U.S.Pat. No. 5,075,109. Delivery systems also include non-polymer systemsthat are: lipids including sterols such as cholesterol, cholesterolesters and fatty acids or neutral fats such as mono- di- andtri-glycerides; hydrogel release systems; sylastic systems; peptidebased systems; wax coatings; compressed tablets using conventionalbinders and excipients; partially fused implants; and the like. Specificexamples include, but are not limited to: (a) erosional systems in whichthe active compound is contained in a form within a matrix such as thosedescribed in U.S. Pat. Nos. 4,452,775, 4,667,014, 4,748,034 and5,239,660 and (b) diffusional systems in which an active componentpermeates at a controlled rate from a polymer such as described in U.S.Pat. Nos. 3,832,253, and 3,854,480. In addition, pump-based hardwaredelivery systems can be used, some of which are adapted forimplantation.

[0053] Use of a long-term sustained release implant may be desirable.Long-term release, are used herein, means that the implant isconstructed and arranged to delivery therapeutic levels of the activeingredient for at least 30 days, and preferably 60 days. Long-termsustained release implants are well-known to those of ordinary skill inthe art and include some of the release systems described above.

[0054] In one aspect of the invention, the cytokine is “co-administered”with a metallocorrinoid which means administered substantiallysimultaneously with a metallocorrinoid. By substantially simultaneously,it is meant that the cytokine (e.g. interferon interferon-β) isadministered to the subject close enough in time with the administrationof the other agent (e.g., vitamin B₁₂ or a cobalamin conjugate), wherebythe two compounds may exert an additive or even synergistic effect.

[0055] The following is provided as an illustration of the presentinvention as it applies to both in vivo and in vitro. The materials,methods, examples, results, and discussions should in no way be viewedas a limitation thereto. For simplicity, the materials and methodssections is provided after the following detailed discussion of thepresent invention.

[0056] Examples, Results and Discussion

[0057]FIG. 1 illustrates NIH-OVCAR-3 ovarian carcinoma evaluated inaccordance with the present invention after 72-hrs growth. Cytokines,particularly interferons, appear to enhance the activity or upregulatethe cellular receptor for vitamin B₁₂ (TCII-R), resulting in enhancedTCII-R activity (in this case demonstrated by NO-Cbl uptake). Singleagent and combination drug effects were assessed to determine whetherIFN-β enhanced NO-Cbl activity. As shown in FIG. 1, NIH-OVCAR-3 cellswere treated continuously with varying concentrations of NO-Cbl andIFN-β. Consistent with our hypothesis, we observed synergisticanti-proliferative activity between IFN-β and NO-Cbl. These matters areshown in the median effect analysis shown in FIG. 2 (similar toisobologram analysis) indicated synergy (a combination index<1) betweenNO-Cbl and IFN-β at all 3 doses tested. Cytotoxicity was noted at thehighest combination dose.

[0058] To assess the effect of IFNβ on TCII-R expression, a western blotanalysis was performed on extracts from NIH-OVCAR-3 cells (ovariancarcinoma) as shown in FIG. 3. Lane 1 is untreated. Lanes 2 and 3 areIFN β treated (200 u/ml) at 4 and 16 hrs respectively. Lanes 4 and 5 areliver and kidney extracts respectively, and serve as a positive control,since TCII-R is abundant in these tissues. As shown in FIG. 3, IFN βcauses an increase in the expression of the TCII receptor, identified asthe monomer at 62 kDa with the corresponding dimer at 124 kDa,consistent with TCII-R. These results correlate with theanti-proliferative effect of co-treatment of NIH-OVCAR-3 cells with IFNβ and nitrosylcobalamin shown in FIG. 1. The increased expression of theTCII receptor by IFN β treatment results in the increased uptake ofnitrosylcobalamin and thus enhanced destruction of the cells. Theco-delivery of IFN-β and nitrosylcobalamin appears to result insynergistic destruction of tumor cells as a result of increased TCIIreceptor expression or activity.

[0059] A flow cytometric analysis of Annexin V positive cells wasperformed to assess the % apoptosis (programmed cell death) ofNIH-OVCAR-3 cells treated with NO-Cbl, alone and in combination withIFN-β. This is illustrated in FIG. 4. The ID25 was used for both NO-Cbl(10 uM) and IFN-β (20 U/mL) for 48 hrs. The effects of IL-2 (250 U/mL)were protective against the effects of NO-Cbl.

[0060] To further elucidate IFN-β upregulated TCII-R, human NIH-OVCAR-3tumors were grown in nude mice to a size of 3 mm in diameter. Thecontrol group received PBS and the treated group received human IFN-β10⁵ units daily for three days. Tumors were harvested, paraffinembedded, and sections were stained with rabbit polyclonal anti-TCII-Rantibody, (provided by Dr. Seetharam's lab, Medical College ofWisconsin). FIG. 5 depicts these treatments. The left panel is anuntreated tumor whereas the right panel is a tumor from a mouse thatreceived IFN-β. The areas stained brown represent TCII-R. A comparisonof the panels demonstrates increased expression of TCII-R with IFN βtreatment. The increased expression of the TCII receptor allows forincreased uptake of NO-Cbl, consistent with the synergy observed in theSRB and Annexin V assays upon NO-Cbl co-treatment with IFN-β.

[0061] WM9 human melanoma was evaluated after 4 days growth. This isshown in FIG. 6. WM9 cells were treated continuously with varyingconcentrations of NO-Cbl and IFN-β. Similar to the NIH-OVCAR-3 cells,there was synergistic anti-proliferative activity between IFN-β andNO-Cbl, as is shown in FIG. 7. Median effect analysis indicated synergy(a combination index <1) between NO-Cbl and IFN-β at all 3 doses tested.

[0062] To further elucidate whether IFN-β upregulated TCII-R, human WM9tumors were grown in nude mice to a size of 3 mm in diameter. Thecontrol group received PBS and the treated group received human IFN-β10⁵ units daily for three days. Tumors were harvested, paraffinembedded, and sections were stained with rabbit polyclonal anti-TCII-Rantibody, (provided by Dr. Seetharam's lab, Medical College ofWisconsin). FIG. 8 depicts these treatments. The upper two panels areuntreated tumors whereas the lower panels are tumors from mice thatreceived IFN-β. The areas stained brown represent TCII-R. A comparisonof the panels demonstrates increased expression of TCII-R with IFN βtreatment.

[0063] One can see from the basal TCIIr activity in the NIH-OVCAR-3 andWM9 stained sections that when TCII-R expression is lower, NO-Cbl uptakeis not pronounced. This is reflected by a higher ID₅₀ associated withthe WM9 cells compared to NIH-OVCAR-3 tumors. Although interferonadministration in both NIH-OVCAR-3 and WM9 resulted in increasedeffectiveness of NO-Cbl, lower basal TCIIR expression in WM9 rendersthese cells less sensitive to the effect of NO-Cbl and the combinationwith IFN-β.

[0064] TCII-R is an important component of metallocorrinoid (e.g.vitamin B₁₂) metabolism and represents a site-specific target toregulate vitamin B₁₂ uptake. Nitrosylcobalamin, a vitamin B₁₂ basedcarrier of nitric oxide (NO), was used to validate the in vivofunctional relevance of increased TCII-R expression. IntraperitonealNO-Cbl treatment of established subcutaneous NIH-OVCAR-3 tumors resultedin tumor regression. The mean volume of untreated tumors was 18 foldgreater compared to NO-Cbl treated tumors at the end of the study.Treated tumors decreased 4-fold in volume during the treatment period.There was no histologic evidence of toxicity to normal tissues at NO-Cbldoses of 170 mg/kg/day after 60 days. IFN-β treatment of NIH-OVCAR-3cells in culture resulted in increased expression of the TCII-R,detected as a monomer (62 kDa) and a dimer (124 kDa). Similarly,immunohistochemical analysis of NIH-OVCAR-3 xenografts from nude micethat received human IFN-β showed increased TCII-R expression compared tocontrols. Tumors that were resistant to IFN-β and NO-Cbl in vivoexhibited minimal to no immunohistochemical evidence of TCII-Rupregulation. In culture, combination treatment with IFN-β and NO-Cblresulted in synergistic anti-proliferative activity in NIH-OVCAR-3 cellsand several different human cells lines including MCF-7 (breast), DU145and LNCap (prostate), ACHN (renal), A549 (lung), WM9, WM35, WM164, andWM3211 (melanoma). Treatment of NIH-OVCAR-3 cells with the combinationof NO-Cbl and IFN-β resulted in a 2-fold increase in annexin V positivecells compared to NO-Cbl alone. Interestingly, a RibonucleotideProtection Assay revealed a ten-fold increase in TRAIL and Caspase 7 inNIH-OVCAR-3 cells treated with the combination of NO-Cbl and IFN-β.Therefore, up-regulation and/or increased activity of the TCII-R byIFN-β results in synergistic anti-tumor effects in vitro and in vivo.

[0065] Materials and Methods

[0066] In-vivo IFN-β treatment of nude mice inoculated with tumors (e.g.WM9—human melanoma or NIH-OVCAR-3—ovarian carcinoma) andImmunohistochemical analysis: Nude mice (n=2 each group), wereinoculated with tumors (e.g. WM9—human melanoma or NIH-OVCAR-3—ovariancarcinoma), subcutaneously (s.c.), one tumor on each flank. The tumorswere grown until 3-5 mm in diameter. Human IFN-β (10⁵ units) wasadministered s.c. for three days to the treatment animals. On day four,animals were sacrificed and tumors were fixed in formalin and paraffinembedded. The sections were analyzed using standard immunohistochemicaltechniques. Anti-TCII-R was used as the primary antibody.

[0067] SRB Anti-proliferative Cell Survival Assay

[0068] Cells (2×10³) were seeded in 96-well plates. Data pointsrepresent mean of eight replicates. (n=8). A control plate was fixed 4hr after seeding (to allow cells to attach) to determine the initialseeding density (A_(ini)). This was defined as 0% growth. To the wellsof the seeded experimental plate, IFN-β was added and incubationcontinued for 3-5 days. Untreated cell controls were included. Growthobtained with this control was defined as 100% (A_(fin)). To determinecell number, cells were fixed with 10% trichloroacetic acid at 4° C. for1 h. They were stained with 0.4% sulforhodamine B prepared in 1% aceticacid at 25° C. for 1 h (27). The wells were washed with 1% acetic acid.Bound dye was eluted with 100 μl of 10 mM Tris-HCl, pH 10.5 andquantitated in a microplate reader at 570 nm. Growth in IFN-β-treatedwells (experimental=exp) was expressed as a percentage of untreatedcontrol growth (mean±SEM).

% Control growth=100%×(A _(exp) −A _(ini))/(A _(fin) −A _(ini))

% STD=100%×(STD _(exp)/(A _(fin) −A _(ini)))

% SEM=100%×(SEM _(exp)/(A _(fin) −A _(ini))) where SEM=STD/{squareroot}n

[0069] Western Blot TCII Receptor

[0070] Cells in culture were treated with vehicle (untreated) or withIFN-β (500 U/ml) for 4 and 16 hrs, washed twice in PBS, harvested byscraping, and lysed in buffer containing 100 mM saline-TRIS. Total cellextracts were homogenized prior to loading. Protein amounts in clarifiedcell extracts were determined using Bio-Rad protein assay reagent.Equivalent amounts of protein (100 μg) were loaded on 10% polyacrylamideSDS separating gels and electrophoresis was performed using glycine-SDSbuffer. Following electrophoresis, gels were equilibrated in transferbuffer 30 min at 25° C., and proteins transferred to nitrocellulosemembrane.

[0071] Immunoblot with Eectro-chemiluminescense Detection

[0072] All steps were performed at 25° C. Following 90 minelectrophoretic wet transfer, the membranes were incubated in washingbuffer TBS-Tween (1×TBS, 0.2% X-100,)+4% BSA for 1-2 hr to blocknon-specific binding. The membrane was washed in washing buffer.Membranes were then incubated in 25 ml of primary antibody at 1:500dilution in the washing buffer overnight at 4° C. Membranes were thenwashed using the washing buffer four times, 10 min each. Membranes wereincubated in 50 ml horseradish peroxidase-conjugated secondary antibody(Zymed) at 1:10,000 dilution in washing buffer for 30 minutes. Membraneswere washed in the washing buffer for two hours. Equal volumes ofelectro-chemiluminescense (ECL) reagents A and B (Amersham) were mixedto give enough reagents to develop the blot (0.125 ml/cm²). Excessbuffer was drained from the membrane and it was placed protein side upon plastic wrap. Detection reagent was added to the protein side of themembrane. The reaction was allowed to continue for exactly 1 minute.Excess detection reagent was drained and the membrane was placed proteinside down on plastic wrap and exposed to film for empirically determinedlengths of time.

[0073] All of the compositions and methods disclosed and claimed hereincan be made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe composition, methods, and in the steps or in the sequence of stepsof the method described herein, without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents that are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

[0074] The following references, to the extent that they provideexemplary procedural or other details supplementary to those set forthherein, are specifically incorporated herein by reference:

[0075] McLean G R, Williams M J, Woodhouse C S, Ziltener H J:Transcobalamin II and in vitro proliferation of leukemic cells. LeukLymphoma 30: 101-9, 1998.

[0076] Tsao C S, Myashita K: Influence of cobalamin on the survival ofmice bearing ascites tumor. Pathobiology 61: 104-8, 1993.

[0077] Jensen H S, Gimsing P, Pedersen F, Hippe E: Transcobalamin II asan indicator of activity in metastatic renal adenocarcinoma. Cancer 52:1700-4, 1983.

[0078] Tsao C S, Miyashita K, Young M: Cytotoxic activity of cobalaminin cultured malignant and nonmalignant cells. Pathobiology 58: 292-6,1990.

[0079] Shimizu N, Hamazoe R, Kanayama H, Maeta M, Koga S: Experimentalstudy of antitumor effect of methyl-B₁₂. Oncology 44: 169-73, 1987.

[0080] McLean G R, Pathare P M, Wilbur D S, Morgan A C, Woodhouse C S,Schrader J W, Ziltener H J: Cobalamin analogues modulate the growth ofleukemia cells in vitro. Cancer Res 57: 4015-22, 1997.

[0081] Huennekens F M, DiGirolamo P M, Fujii K, Jacobsen D W, Vitols KS: B12-dependent methionine synthetase as a potential target for cancerchemotherapy. Adv Enzyme Regul 14: 187-205, 1976.

[0082] Bauer, Joseph A., Characterization and nitric oxide releaseprofile of nitrosylcobalamin: a potential chemotherapeutic agent.Anti-Cancer Drugs 1998; 9(3): 239-244.

1. The therapeutic composition comprising a cobalamin drug conjugate anda cytokine.
 2. The composition of claim 1, wherein the cobalamin drugconjugate is a vitamin B₁₂ analog.
 3. The therapeutic composition ofclaim 1, wherein the cobalamin drug conjugate is selected from the groupconsisting of hydroxocobalamin, cyanocobalamin, methylcobalamin,5′deoxyadenocobalamin, nitrosylcobalamin, and radiolabeled vitamin B₁₂analog.
 4. The therapeutic composition of claim 1, further including apharmaceutical carrier.
 5. The therapeutic composition of claim 1,wherein the cytokine is an interferon-α.
 6. A method of enhancing uptakeof a cobalamin drug conjugate comprised of administering a cytokine andadministering a cobalamin drug conjugate.
 7. The method of claim 6,wherein said cobalamin the cobalamin drug conjugate is a vitamin B₁₂analog.
 8. The method of claim 6, wherein the cobalamin drug conjugateis selected from the group consisting of hydroxocobalamin,cyanocobalamin, methylcobalamin, 5′deoxyadenocobalamin,nitrosylcobalamin, and radiolabeled vitamin B₁₂ analog.
 9. The method ofclaim 6, wherein the cytokine is administered with a pharmaceuticalcarrier.
 10. The method of claim 6, wherein said cytokine is aninterferon-α.
 11. A method of increasing TCII-R activity in a subject totreat a condition favorably affected by an increase in said TCII-Ractivity comprising the step of administering to a subject in need ofsuch treatment a cytokine in an amount effective to increase TCII-Ractivity in the subject.
 12. The method of claim 11, further comprisingthe step of co-administering a cobalamin drug conjugate.
 13. The methodof claim 12, wherein the cobalamin drug conjugate is a vitamin B₁₂analog.
 14. The method of claim 12, wherein the cobalamin drug conjugateis selected from the group consisting of hydroxocobalamin,cyanocobalamin, methylcobalamin, 5′deoxyadenocobalamin,nitrosylcobalamin, and radiolabeled vitamin B₁₂ analog.
 15. The methodof claim 12, wherein said cytokine is administered prior to thecobalamin drug conjugate.
 16. The method of claim 12, wherein saidcytokine is administered prophylactically.
 17. The method of claim 12,wherein said cytokine is administered acutely.
 18. The method of claim11, wherein said cytokine is an interferon-α.
 19. The method of claim11, wherein said condition is unwanted cellular proliferation.
 20. Themethod of claim 11, wherein the cytokine is administered with apharmaceutical carrier.